The Influenza A virus (IAV) RNA-dependent RNA polymerase (RdRp) is comprised of three proteins, the polymerase acid (PA), polymerase basic 1 (PB1) and polymerase basic 2 (PB2) proteins. Although there is no crystal structure of this large ~250 kDa complex, crystal structures have been obtained for several domains of the polymerase such as the PA C-terminal domain (PA-CTD) in complex with a short peptide derived from the PB1 protein (He, X. et al. Nature, 2008, 454, 1123 and Obayashi, E. et al. Nature2008, 454, 1127). SSGCID obtained the first crystal structures of the PA C-terminal domain (PA-CTD) in the absence of a peptide derived from PB1 (1933 Wilson-Smith human H1N1 PDB ID 4IUJ, SSGCID-InvaN.07057.a at 1.95 Å resolution and 2013 Anhui avian H7N9 PDB ID 4P9A, SSGCID-InvaQ.07057.a at 2.25 Å resolution). In comparison with the PB1 peptide-bound structures, the apo SSGCID PA structures revealed a flexible PB1 binding site with many residues which are disordered or form different interactions in the apo state than that observed in the PB1 bound state. Such plasticity may allow for different binding partners in this region.

The influenza virus polymerase complex comprised of PA-PB1-PB2 is believed to be a drug target of significant interest, and a few groups have published efforts to obtain peptide inhibitors (Wunderlich K. et al. Antimicrob Agents Chemother., 2011, 55, 696) or small molecule inhibitors identified with mM affinity by virtual screening (Muratore, G. et al. PNAS, 2012, 109, 6247; Tintori, C. et al. BMCL, 2014, 24, 280; Pagano M. et al. ChemMedChem, 2014, 9, 129). As part of a community research project (CRP60) we performed fragment screening using saturation transfer difference NMR spectroscopy (Begley, D.W. et al. Curr Protoc Chem Biol.2013, 5, 251) on the H1N1 PA-CTD protein and obtained 39 confirmed fragment hits. The primary collaborator (Brian Hubbard) and his former industry colleagues (Michael Serrano-Wu and Virendar Kausik) would like to continue to collaborate with SSGCID to fully characterize the 39 confirmed hits as well as 4 commercially available compounds described in the references above and to begin the process of elaborating these compounds into novel chemical matter to target influenza A viruses.

Specific Aim #1: Initial Fragment Hit Characterization.Work to be performed at Emerald Bio and third party collaborator (SPR), carried out during Q(s) Q1-Q2.

We plan to use a variety of techniques to characterize the 39 fragment hits identified by STD-NMR. Differential scanning fluorimetry (DSF) analysis of H1N1 and N7N9 PA-CTD showed a +14-16 °C shift in the presence of PB1 peptide and a more complex but overall similar thermal stabilization in the presence of one of the compounds reported in the literature. We will perform thermal shift analysis to determine the degree of thermal stabilization for each of the 39 fragment hits. Additional NMR experiments (STD, WLG, HSQC) will be performed based off STD-NMR and thermal shift rank ordering to determine if the fragments are competitive or non-competitive with the PB1-derived peptide. These experiments will be performed at Emerald. The top 10 hits from these experiments will go into surface plasmon resonance (SPR) characterization of binding affinity and on and off rates, which will be performed by the requestor or his collaborators outside SSGCID.

Specific Aim # 2: Co-crystal structure determination of PA-CTD with fragments and literature inhibitors.Work to be performed at Emerald Bio and collaborator, carried out during Q(s) Q2-Q3.

Our initial apo crystals structures of H1N1 and H7N9 PA-CTD contain significant disorder in part of the PB1 binding site as well as other portions that are occluded due to crystal lattice contacts. However, the main binding surface (Q408-F411-N412-C415-K643-W706) is accessible to solvent in the current crystal form. Therefore, we will attempt both soaking experiments as well as co-crystallization to obtain new crystal forms using both of the current H1N1 and H7N9 PA-CTD constructs as well as versions of those proteins without the affinity tag removed. These crystal structures will be used to rationally design the next round of compounds. Co-crystallization studies will be performed at Emerald and the rational design will be performed by the collaborator and/or his collaborators outside SSGCID.

Specific Aim 3: Chemical elaboration of initial hits.Work to be performed at Emerald Bio and collaborator, carried out during Q(s) Q2-Q4.

Given the compounds reported in the literature as well as the 39 hits identified by STD-NMR, we start to elaborate these hits using catalog SAR and design and synthesis of new compounds. The catalog SAR and design will take place collaboratively, but largely by the collaborator. Chemical synthesis of the collaborator designed compounds will likely be performed by a third party. Once these new compounds arrive at Emerald, they will be characterized by a panel of biophysical binding experiments (STD-NMR, DSF) and co-crystallization at Emerald. At this point, we should have enough data on a few different chemical series for publication in a high impact journal.

The results will be used to generate and qualify lead chemical matter targeting the influenza PA-PB1 interface. The deliverables will be qualification experimental results (DSF, STD-NMR and other NMR competition experiments), co-crystal structures which will be deposited into the Protein Data Bank, and ultimately a manuscript detailing the experimental results and conclusions.

Given the high-throughput nature of the DSF, STD-NMR and co-crystallization experiments we project approximately 400 hours at Emerald Bio for this research. Additional funds will be necessary for catalog SAR as well as chemical synthesis (by HBS or third party collaborator) and SPR analysis (by HBS or third party collaborator).

* not necessary due to a large number of available analog by catalog compounds targeting milestones 4 and 7

Summary: The primary goal of the functional study is to characterize and improve upon fragment hits obtained against the polymerase acidic protein C-terminal domain (PA-CTD) of the influenza virus RNA-dependent RNA polymerase. These hits were identified under a community research project (CRP_60) during Sept 2013-August 2014. The apo crystal structures of H1N1 (InvaN.07057.a, PDB ID 4IUJ) and H7N9 (InvaQ.07057.a, PDB ID 4P9A) were published previously (Moen, S.O. et al. Sci. Rep. 2014, 4, 5944). During the first half year of the functional study, starting Sept 2014, we achieved milestones 1, 2, 4, 5, and 7 directed toward all three specific aims. During the second half of the year we achieved milestone 3 and also performed second and third rounds of SAR which replicated milestone 4 and 7, making milestones 6 and 8 unnecessary (chemical synthesis). We obtained a total of 9 crystal structures of PA-CTD with fragment compounds (6 in the first half of the year and 3 in the second half). These results are going into a manuscript to be submitted in the next few months. In addition, we have spoken with a collaborator (Yoshi Kawaoka, University of Wisconson-Madison) who would be willing to perform cellular assays to see if any of these fragment-sized molecules possess cellular activity. These results will also go into the manuscript.

Completed. Although we completed this Specific Aim during the first half of the year, we continued to try to obtain additional co-crystal structures with fragments identified from the original screen and follow on confirmation experiments. Most of our effort targeted compounds which did not contain the main chlorophenyl chemical scaffold, but unfortunately were not successful. Nevertheless, we obtained a co-crystal structure with one additional compound from the original screen, EBSI-39, which may be the smallest fragment co-crystal structure obtained for the project.

Specific Aim 3: Chemical elaboration of initial hits.

Completed. We analyzed over 20 follow on compounds through 3 rounds of acquisition and analysis. During the first half of the year we obtained co-crystal structures with three of these (EBSI-4719, EBSI-4720, and EBSI-4721). During the second half of the year we obtained only a single co-crystal structure with EBSI-4723 and that one with only partial occupancy. Nevertheless, we obtained biophysical data on quite a number of follow on compounds including % max STD-NMR. For the series below, the % max STD-NMR showed a clear improvement in binding from F- to Cl- to Br- which mirrored the binding observed crytallographically. Replacing the chlorophenyl with a chlorothiophene (EBSI-4723) increase binding significantly.

Fragments 576 and 7474 were combined into fragment 4719, for which we obtained a 2.45 Å resolution data set with improved binding as shown by %max STD-NMR. These compounds were further elaborated with a number of compounds (EBSI-4849, 5103, 5103, 5507, 5508). Interestingly, the FDA approved asthma drug Montelukast (Singulair) as well as the related compound Verlukast (MK-571) also fit this same profile. These compounds alongwith 5507 and 5508 were obtained in the last round of analog by catalog and were analyzed by STD-NMR and crystallographic soaking experiments as the functional study was coming to a close.